Bna.EPF2 Enhances Drought Tolerance by Regulating Stomatal Development and Stomatal Size in Brassica napus

Drought stress severely affects global plant growth and production. The enhancement of plant water-use efficiency (WUE) and drought tolerance by the manipulation of the stomata is an effective strategy to deal with water shortage. However, increasing the WUE and drought tolerance by manipulation on the stomata has rarely been tested in Brassica napus. Here, we isolated Bna.EPF2, an epidermal patterning factor (EPF) in Brassica napus (ecotype Westar), and identified its role in drought performance. Bna.EPF2 overexpression lines had a reduction average of 19.02% in abaxial stomatal density and smaller stomatal pore size, leading to approximately 25% lower transpiration, which finally resulted in greater instantaneous WUE and enhanced drought tolerance. Interestingly, the reduction in stomatal density did not affect the CO2 assimilation or yield-related agronomic traits in Bna.EPF2 overexpression plants. Together with the complementation of Bna.EPF2 significantly decreasing the stomatal density of Arabidopsis epf2, and Bna.EPF2 being expressed in mature guard cells, these results suggest that Bna.EPF2 not only functions in stomatal density development, but also in stomatal dimension in Brassicas. Taken together, our results suggest that Bna.EPF2 improves WUE and drought tolerance by the regulation of stomatal density and stomatal size in Brassica without growth and yield penalty, and provide insight into the manipulation of this gene in the breeding of drought tolerant plants with increased production under water deficit conditions.


Introduction
Drought stress is one of the most destructive environmental stresses to plants. It severely affects plant development and growth, and becomes a key factor limiting plant productivity [1,2]. The stomata function as key valves that regulate the gas exchange between plants and the environment, by uptaking CO 2 for photosynthesis and losing water for transpiration. Reducing transpiration rates via the stomata is efficient for most plants under water deficit, and they thus increase their water use efficiency (WUE) [3]. Therefore, reducing transpiration and enhancing the WUE of plants under drought stresses without yield penalties has become the main objective for plant breeding programs.
Plants can regulate transpiration and WUE by managing stomatal conductance, which is mainly determined by stomatal density and stomatal movement [4][5][6][7][8][9]. For short-term water deficit, plants close the stomata to reduce transpiration and improve WUE. Under long-term water deficit conditions, plants produce maximum stomatal conductance by changing the stomatal size and density [10,11]. Therefore, manipulating the stomata number is one of the ways to alter the photosynthetic efficiency and water transpiration of plants [12][13][14], thus improving plant physiological characters and providing the potential for increasing crop yield. 2 of 16 In dicots, stomatal development begins with an asymmetric division of the meristematic mother cell (MMC), which generates a small triangular-shaped meristemoid (M) and a large stomatal lineage ground cell (SLGC). The SLGC differentiates into an epidermal pavement cell or another meristemoid, whereas an M then differentiates into a guard mother cell (GMC). Finally, the GMC divides symmetrically to form a pair of guard cells (GC) [14]. EPFs polypeptide hormones, encoded by EPIDERMAL PATTERNING FAC-TOR family members, are secreted as an extracellular signal for stomatal development initiation, and the receptors TMM (too many mouths) and ERECTA in the surrounding cells perceive these extracellular signals [15][16][17], and then transfer the signals across the membrane to intracellular components by the MAPK signaling pathway to initiate or inhibit stomatal development [18][19][20][21]. There are a total of 11 members of the EPF family in Arabidopsis, including EPF1, EPF2, and nine EPFLs [20][21][22][23]. Four members of the EPF family, EPF1, EPF2, EPFL9, and EPFL6, were found to participate in the process of stomatal development [23][24][25][26][27][28]. EPF1, EPF2, and EPFL6 are negative regulators, and EPFL9 is a positive regulator of stomatal density by primarily controlling the initiation and frequency of asymmetric cell divisions and finally determining the occurrence and density of stomata in Arabidopsis [20,23,26,29]. EPFL9, also named STOMAGEN, regulates stomatal development by antagonizing EPF1 and EPF2 [27,28,30]. Due to the roles of EPFs in stomatal development, their effects on water relations and net photosynthesis have been tested in Arabidopsis, crops, and trees. The overexpression of EPF2 significantly improved drought tolerance without deleterious effects on the nutrient uptake in Arabidopsis [12,31]. The ectopic overexpression of PdEPF2 enhanced drought tolerance by modulating stomatal density and regulated the ABA response of Arabidopsis transgenic plants [32], and the ectopic overexpression of MdEPF2 from apple improved WUE and reduced oxidative stress in tomato [33]. The overexpression of EPF1 homologs improved plant WUE and drought tolerance by reducing stomatal density in poplar and wheat [34,35]. The overexpression of OsEPF1 in rice plants showed reduced stomatal density and increased root cortical aerenchyma formation [36], and HvEPF1 overexpression in barley caused significantly enhanced WUE and drought tolerance [29]. The genetic engineering of EPFL9/Stomagen or the spraying of stomagen peptide led to high stomatal densities and crop production, in which the photosynthetic rate was enhanced by an average of 30% [12,28,37]. EPFL family genes exist in many land plants. However, no such reports have been reported in Brassica, and whether the functions of the EPFL family in the dicotyledon Brassica are also conserved remains unclear.
Brassica napus is a major oilseed crop in China and approximate 20% of the world's yield is produced by China [38]. However, the high productivity and quality strongly depends upon water availability [39], and the trade-offs between growth and stress resistance in plants has long been a research focus for scientists and research. Although most previous studies of EPF2 on both herbaceous and woody plants suggested vital roles in regulating stomatal development, whether it performs the same function in Brassica napus needs further experimental evidence. In this study, we focused on the homologous gene of EPF2 in Brassica napus and identified its role in stomata development and the effect on water use efficiency, drought tolerance, and yield traits. The expression of Bna.EPF2 clearly decreased stomatal density in Brassica napus and restored the stomatal phenotype of Arabidopsis epf2 mutant plants. The Bna.EPF2 overexpression plants had higher WUE, lower transpiration rate and stomatal conductance at normal growth conditions and increased drought tolerance compared to wild-type plants under water limited conditions. Interestingly, the seed yield per plant was not affected in these overexpression plants. This study revealed the conserved role of Bna.EPF2 in stomatal development, and a new role in stomatal dimension compared to other EPF2 orthologs was reported which coordinated with the water use efficiency of plants. Our results suggested the potential of Bna.EPF2 in crop drought tolerance improvement and seed yield per plant and provided a new gene resource for genetic manipulation.

Isolation and Characterization of Bna.EPF2 in Brassica napus
To identify orthologs of EPF and EPFL family members in Brassica napus, all 11 previously reported EPF and EPFL peptides in Arabidopsis were used as a BLASTP query to search the Brassica napus genome (http://plants.ensembl.org/Brassica_napus, AST_PRJEB5043_v1, accessed on 1 September 2014). A total of 25 putative EPF/EPFLs were obtained. A phylogenetic tree of these predicted Bna.EPFL peptides with those from Arabidopsis were constructed using MEGA 6.06 and the maximum likelihood method ( Figure 1A). Each Arabidopsis EPF/EPFL had 1-2 orthologs in B. napus, in A and/or C genomes. EPF1, EPF2, EPFL9, and EPFL6 were reported to be involved in stomatal development in Arabidopsis [20,23,26]. As shown in Figure 1A, there were two closed Arabidopsis EPF1 orthologs in B. napus, one from the A genome (BnaA09g43550D) and the other from the C genome (BnaC08g36150D). EPFL6 and EPFL9 also had two orthologs in B. napus, respectively, with one each in the A and C genomes ( Figure 1A), whereas only one closed ortholog of EPF2, BnaA09g23140D, was presented in the B. napus genome and, therefore, it was named Bna.EPF2 in this study. Two orthologs of EPF1, EPFL6, and EFPL9 in Brassica showed high sequence similarity (Supplemental Figure S1).
Bna.EPF2 had 119 amino acids (AAs), including a 25AA long signal peptide, and a 49AA EPF domain (https://smart.embl-heidelberg.de, 3 March 2023, version 9), with 13.17 KD molecular weight and 9.10 isoelectric point (https://web.expasy.org/cgi-bin/ protparam/protparam, 3 March 2023). Phylogenetic analysis and the multiple sequence alignment of EPF2s in both dicots (Brassica napus, Arabidopsis thaliana, and Glycine max) and monocots (Sorghum bicolor, Zea mays, and Oryza sativa) indicated that Bna.EPF2 was the most closed ortholog of AtEPF2, with an 81.67% protein identity ( Figure 1B). These results indicated that Bna.EPF2 may have a similar function in stomatal development to AtEPF2. EPFs have a signal peptide at the N-terminus, which needs to be cleaved before they function. Through a comparison of the sequence of Bna.EPF2 with other orthologs and the prediction of the cleavage site as reported in AtEPF2 [40], the mature peptide of Bna.EPF2 was found to be 45AA in length, and there was only 1 amino acid different from AtEPF2 ( Figure 1B). Therefore, we chose Bna.EPF2 for further functional identification.

Expression Pattern of Bna.EPF2
To examine the expression pattern of Bna.EPF2, we generated transgenic lines expressing the β-glucuronidase (GUS) reporter gene under the control of the promoter of the Bna.EPF2 gene ( Figure 2A). Histochemical GUS staining showed a high expression of Bna.EPF2 in young leaves, but not in roots or stems at the seedling stage. Bna.EPF2 was also expressed in flowers and siliques at the reproductive stage, with a higher expression in calyx, ovary, and stigma, but not in petals and stamens ( Figure 2B). At the cellular level, Bna.EPF2 likely had a preferential expression in the stomatal lineage cells of leaves and calyx, with a great expression in mature guard cells ( Figure 2C). In contrast to Arabidopsis EPF2, Bna.EPF2 was only expressed in stomatal precursor cells, and enriched in the early meristemoid mother cell (MMC) [23]. These results revealed that Bna.EPF2 had a similar but expanded expression pattern compared to the AtEPF2 gene in Arabidopsis, indicating that Bna.EPF2 may have a broader function than AtEPF2.

The Ectopic Expression of Bna.EPF2 Rescued the Stomatal Development Defects in Arabidopsis epf2 Mutant
The mutation of Arabidopsis EPF2 caused increased stomatal density, and its overexpression plants showed a phenotype of reduced stomatal density [12]. To explore whether Bna.EPF2 is also involved in stomatal development and has a similar function to AtEFP2, we expressed Bna.EPF2 CDS driven by 35S promoter in the Arabidopsis epf2 mutant plants, in which the stomatal density was increased as reported ( Figure 3A) [26]. We found that the expression of Bna.EPF2 dramatically reduced the stomatal density of epf2 ( Figure 3B). Statistical analyses showed that these three randomly selected independent lines had an even lower level of stomatal density than Col-0, with an average of 13.04-20.49% decline compared to Col-0 ( Figure 3A,B). Stomatal index, the ratio of stomata to all epidermal cells (pavement cells and stomata) in a given area (per mm 2 ), a more accurate indicator reflecting stomatal development, was also decreased by 14.04-22.91% in these Bna.EPF2 expression epf2 transgenic lines ( Figure 3C). These data suggested that Bna.EPF2 has a conserved function as AtEPF2, negatively regulating stomatal development.
were obtained. A phylogenetic tree of these predicted Bna.EPFL peptides with those from Arabidopsis were constructed using MEGA 6.06 and the maximum likelihood method ( Figure 1A). Each Arabidopsis EPF/EPFL had 1-2 orthologs in B. napus, in A and/or C genomes. EPF1, EPF2, EPFL9, and EPFL6 were reported to be involved in stomatal development in Arabidopsis [20,23,26]. As shown in Figure 1A, there were two closed Arabidopsis EPF1 orthologs in B. napus, one from the A genome (BnaA09g43550D) and the other from the C genome (BnaC08g36150D). EPFL6 and EPFL9 also had two orthologs in B. napus, respectively, with one each in the A and C genomes ( Figure 1A), whereas only one closed ortholog of EPF2, BnaA09g23140D, was presented in the B. napus genome and, therefore, it was named Bna.EPF2 in this study. Two orthologs of EPF1, EPFL6, and EFPL9 in Brassica showed high sequence similarity (Supplemental Figure S1).

The Ectopic Expression of Bna.EPF2 Rescued the Stomatal Development Defects in Arabidopsis epf2 Mutant
The mutation of Arabidopsis EPF2 caused increased stomatal density, and its overexpression plants showed a phenotype of reduced stomatal density [12]. To explore whether Bna.EPF2 is also involved in stomatal development and has a similar function to AtEFP2, we expressed Bna.EPF2 CDS driven by 35S promoter in the Arabidopsis epf2 mutant plants, in which the stomatal density was increased as reported ( Figure 3A) [26]. We found that the expression of Bna.EPF2 dramatically reduced the stomatal density of epf2 ( Figure 3B). Statistical analyses showed that these three randomly selected independent lines had an even lower level of stomatal density than Col-0, with an average of 13.04-20.49% decline compared to Col-0 ( Figure 3A,B). Stomatal index, the ratio of stomata to all epidermal cells (pavement cells and stomata) in a given area (per mm 2 ), a more accurate indicator reflecting stomatal development, was also decreased by 14.04-22.91% in these Bna.EPF2 expression epf2 transgenic lines ( Figure 3C). These data suggested that Bna.EPF2 has a conserved function as AtEPF2, negatively regulating stomatal development.

Overexpression of Bna.EPF2 in Brassica Plants Caused a Lower Stomatal Density and Smaller Stomatal Complex Size
To further investigate the function of Bna.EPF2, we overexpressed Bna.EPF2 into the B. napus species Westar. Three transgenic lines, OE-17, OE-19, and OE-29, in which Bna.EPF2 was overexpressed, were randomly selected for the determination of stomatal density and index phenotypes ( Figure 4A). Bna.EPF2 overexpression plants exhibited an average of 19.02% lower stomatal density and 6.90% lower stomatal index than WT in true leaves ( Figure 4B-D), as well as an average of 28.96% lower stomatal density and 9.05% lower stomatal index than WT in cotyledons (Supplemental Figure S2), consistent with the role of EPF2 in Arabidopsis. These results further suggested that Bna.EPF2 and AtEPF2 maintained functional conservation in stomatal development.  In addition to the stomatal density changes, we found, interestingly, that the stomatal complex size was also altered in the Bna.EPF2 overexpression plants ( Figure 4E-J). Bna.EPF2 overexpression plants had a smaller stomatal complex size, with reduced complex length and/or width ( Figure 4F,G). Stomatal pore length, pore width, and pore area were also reduced by an average of 6.78%, 4.80% and 10.90% in these transgenic lines ( Figure 4H-J), respectively. Together with the fact that Bna.EPF2 was expressed in young and mature guard cells ( Figure 2C), these results suggested that Bna.EPF2 is also involved in stomatal dimension regulation, in addition to its regulation in stomatal density development. Values are means ± SE (each with at least 60 stomata analyzed per experiment). No significant difference (ns.), p ≥ 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; Student's t-test.
In addition to the stomatal density changes, we found, interestingly, that the stomatal complex size was also altered in the Bna.EPF2 overexpression plants ( Figure 4E-J). Bna.EPF2 overexpression plants had a smaller stomatal complex size, with reduced complex length and/or width ( Figure 4F,G). Stomatal pore length, pore width, and pore area were also reduced by an average of 6.78%, 4.80% and 10.90% in these transgenic lines ( Figure 4H-J), respectively. Together with the fact that Bna.EPF2 was expressed in young and mature guard cells ( Figure 2C), these results suggested that Bna.EPF2 is also involved in stomatal dimension regulation, in addition to its regulation in stomatal density development.

Overexpression of Bna.EPF2 in B. napus-Conferred Enhanced Drought Tolerance
As the stomata control CO 2 influx and the loss of transpiration water, changes in plant transpiration caused by stomatal density and dimension changes will affect the leaf temperature and drought performance. We therefore analyzed the leaf temperature of Bna.EPF2 overexpression lines and WT plants at normal growth conditions. The leaf temperature was 0.23 • C-1 • C higher in the Bna.EPF2 overexpression plants than that of WT plants ( Figure 5A,B), consistent with the reduced stomatal density and stomatal pore area in the Bna.EPF2 overexpression plants ( Figure 4B,J). Next, drought stresses were applied to 14-day-old WT and Bna.EPF2 overexpression plants. During drought stresses, the wild-type plants wilted earlier than the Bna.EPF2 overexpression plants ( Figure 5C,D). When water was resupplied for five days, an average of 70.14% of the Bna.EPF2 overexpression plants were recovered, whereas only about 8.33% of the WT plants were recovered ( Figure 5C,D)

Overexpression of Bna.EPF2 in B. napus-Improved WUE by Reducing Transpiration
To explore the impact of decreased stomatal density and stomatal pore size in Bna.EPF2 overexpression plants, stomatal conductance, leaf transpiration, and inst neous WUE (photosynthesis/transpiration) were analyzed using a Li-6400XT Photo thesis system. The stomatal conductance at the steady state under 450 ppm CO2 condi was reduced by 19.65-33.04% in these transgenic Brassica plants ( Figure 6A), which mainly caused by their lower stomatal density and stomatal pore area ( Figure 4B,J). sistent with the reduced stomatal conductance, the Bna.EPF2 overexpression plants h

Overexpression of Bna.EPF2 in B. napus-Improved WUE by Reducing Transpiration
To explore the impact of decreased stomatal density and stomatal pore size in the Bna.EPF2 overexpression plants, stomatal conductance, leaf transpiration, and instantaneous WUE (photosynthesis/transpiration) were analyzed using a Li-6400XT Photosynthe-sis system. The stomatal conductance at the steady state under 450 ppm CO 2 conditions was reduced by 19.65-33.04% in these transgenic Brassica plants ( Figure 6A), which was mainly caused by their lower stomatal density and stomatal pore area ( Figure 4B,J). Consistent with the reduced stomatal conductance, the Bna.EPF2 overexpression plants had a decrease of 21.57-31.59% in the leaf transpiration rate (Tr) compared to WT plants (Figure 6B), and a significant 3.14-5.49% higher instantaneous WUE (iWUE) (Figure 6C To further determine the effects of stomatal density on leaf gas exchange, CO2 assimilation, leaf transpiration, and instantaneous WUE under a range of external CO2 concentrations and light intensities were also examined. A-Ci curves and light curves showed that the Bna.EPF2 overexpression plants had a similar level of assimilation rate under a range of CO2 concentrations and saturating light levels ( Figure 6D,G). The transpiration EPF2 over-expression lines in response to shifts in light intensity. All data were measured by a Li-6400XT gas exchange system. n ≥ 3 leaves per genotype per experiment, experiments were repeated three times, values are means ± SE, *, p < 0.05; **, p < 0.01; ***, p < 0.001; Student's t-test, asterisk (*) with different colors present the significant difference of the different Bna.EPF2 overexpression lines compared to WT at p < 0.05 level.
To further determine the effects of stomatal density on leaf gas exchange, CO 2 assimilation, leaf transpiration, and instantaneous WUE under a range of external CO 2 concentrations and light intensities were also examined. A-Ci curves and light curves showed that the Bna.EPF2 overexpression plants had a similar level of assimilation rate under a range of CO 2 concentrations and saturating light levels ( Figure 6D,G). The transpiration of Bna.EPF2 overexpression plants was consistently reduced under different CO 2 concentrations and light intensities, with a 17.64-26.5% reduction under different CO 2 conditions and a 13.96-37.54% reduction under different light intensities compared to WT ( Figure 6E,H), matching well with their reduced stomatal density and dimension (Figure 4). The Bna.EPF2 overexpression lines had a comparable or even higher instantaneous WUE than WT in response to the shifts in light intensity or CO 2 concentrations ( Figure 6F,I). These results demonstrated that the overexpression of Bna.EPF2 affected leaf transpiration by regulating stomatal density and dimension in B. napus, further suggesting the potential role of Bna.EPF2 in the improvement of WUE and drought tolerance in plants.

Overexpression of Bna.EPF2 Did Not Influence Major Agronomic Traits
The decreased stomatal density and smaller stomatal pores in the Bna.EPF2 overexpression plants may have affected plant growth and yield under the natural field. To explore this, we measured the agronomic traits of the transgenic Brassica plants that grew in a Wuhan natural field. Bna.EPF2 overexpression had no significant impact on plant height, the length of main inflorescence, the number of siliques on main inflorescence, the number of the first branch, 1000-seed weight, or seed yield per plant, which were all similar to those of WT plants. Interestingly, the length of the silique and number of seeds per silique were significantly increased, with an average increase of 4.46% and 4.02% in the Bna.EPF2 transgenic lines, respectively (Table 1), consistent with the continuous expression of Bna.EPF2 in the ovary and young siliques of transgenic plants ( Figure 2B). These results indicated that the overexpression of Bna.EPF2 reduced stomatal density and dimension without yield penalty. Bna.EPF2 and AtEPF2 have similar functions in stomatal development, indicating that there is a common pathway for stomatal development in both Arabidopsis and B. napus. Three bHLH transcription factors, SPCH, MUTE, and FAMA, are key regulators of stomatal development, from the initial transition of a protodermal cell to mature guard cells in Arabidopsis, and may be conserved from basal land plants through to angiosperms.
In addition, the subtilisin-like serine protease STOMATAL DENSITY AND DISTRIBU-TION1 (SDD1) acts as a negative regulator of stomatal development independent of the main signaling pathway. We searched the SPCH, MUTE, FAMA, and SDD1 homologous genes in B. napus using BLAST alignments (https://plants.ensembl.org/Brassica_ napus, accessed on 3 March 2023), and found that each had multiple copies in B. napus ( Figure S3A-D). Therefore, we selected BnaC02G14500D, BnaA03g57400D, BnaA03g37180D, and BnaA10g02390D as the candidate genes for Bna.SPCH, Bna.MUTE, Bna.FAMA, and Bna.SDD1, respectively, and determined their expression levels in the transgenic plants, since they had the highest protein identities compared with Arabidopsis. The relative expression levels of Bna.SPCH and Bna.MUTE were significantly decreased in Bna.EPF2 overexpression lines compared to WT, and Bna.FAMA was not significantly altered ( Figure S3E), consistent with the phenotypes of decreased stomatal density in the Bna.EPF2 overexpression lines ( Figure 4B), as well as in the Arabidopsis spch and mute mutant plants [40]. Surprisingly, the expression level of Bna.SDD1 was also significantly decreased. These results suggested that Bna.SPCH and Bna.MUTE have similar roles in stomatal development to Arabidopsis, and the overexpression of Bna.EPF2 may affect the stomatal density via the transcriptional inhibition of SPCH and MUTE in different ways, thereby inhibiting the asymmetric cell division and thus regulating stomatal density.

Discussion
Drought stress is one of the major adverse environmental stresses, causing damage to crop development and huge yield loss. As one of the most important oil crops, B. napus ranks first in the proportion of sowing area and total yield in the world. In the context of climate change, environmental stresses and their impact on yield formation are becoming more and more prominent. It is a big challenge in crop breeding to identify genes that confer drought tolerance, but have no significant penalty on yield. The stomata function as valves for gas exchange, therefore, manipulating the stomatal density is a good way for breeding plants to cope with water shortage conditions. Stomatal development has been well studied in Arabidopsis, with many genes having been reported to participate in this process. EPFs/EPFLs are the most important ones that play key roles in stomatal development and distribution [41], and they may represent a potential target for crop improvement [29,[31][32][33][34]42,43]. Sequence and phylogenetic analyses showed that there was only one closed EPF2 ortholog in the genome of B. napus, named Bna.EPF2 in this study (Figure 1). Based on the findings that Bna.EPF2 expression rescued the increased stomatal density phenotype of Arabidopsis epf2 ( Figure 3B), and that Bna.EPF2 overexpression caused decreased stomatal density and index in Brassica Westar ( Figure 4B,C), these results suggested that Bna.EPF2 was also involved in stomatal development, with a similar function to Arabidopsis EPF2. Changing the expression of EPF2 orthologs in other plants, such as barley, poplar, and apple, also influenced the stomatal development [29,32,33], suggesting that the function of EPF2s in stomatal density is highly conserved in plants. As SPCH and MUTE regulate distinct stomatal developmental steps [44][45][46][47][48], Bna.SPCH and Bna.MUTE were significantly reduced in Bna.EPF2 overexpression Brassica plants ( Figure S3), which coincided with results in Arabidopsis, suggesting that EPF2 peptides control asymmetric cell division in the early stomatal lineage and restrict entry into the stomatal lineage or promote an exit to the pavement cell fate, and thus regulate stomatal density. In contrast to AtEPF2 [26], Bna.EPF2 was also enriched in young and mature guard cells ( Figure 2C) and its overexpression in Brassica caused a smaller stomatal complex size and stomatal pore size (Figure 4). These results suggested that Bna.EPF2 has a broader function in stomatal biology. In addition to its conserved role in stomatal density regulation, it also plays a role in stomatal dimension. The function of EPFs in stomatal dimension has not yet been reported, and therefore the ways in which Bna.EPF2 regulates stomatal dimension can be investigated in the future, which may lead to the exploration of new roles of EPFs in stomatal biology.
The iWUE and drought tolerance were significantly enhanced in Bna.EPF2 overexpression transgenic plants ( Figures 5C and 6C), in line with a reduction in the stomatal density and stomatal pore area ( Figure 4B,J). These results suggested that the manipulation of stomatal development in Brassica improves WUE and drought tolerance. The gas exchange is controlled by stomata and influenced by stomatal movements (opening and closing), stomatal density, and pore size [49][50][51][52]. The CO 2 assimilation rate, stomatal conductance, and transpiration rate of plants change significantly under abiotic stresses [53,54], indicating that the reduction in stomatal conductance may affect CO 2 assimilation rates and limit the production of biomass due to the limitations of gas exchange [55]. However, recent reports have showed that a small reduction in the stomatal density did not has a significant impact on the biomass or yield. For example, Arabidopsis EPF2 overexpression plants that had an 80% reduction in stomatal density showed only a small reduction in net photosynthesis at high light, EPFL9-RNAi plants with a 68% reduction in stomatal density showed no significant differences in photosynthesis compared to Col-0 plants [42,56], and HvEPF1 overexpression barley plants had a 58% reduction in stomatal density and showed no reductions in seed yield [29]. Additionally, the overexpression of both HARDY and HDG11, and mutations of GTL1 and GPA1, reduced stomatal density and improved drought tolerance with no penalty on carbon assimilation or biomass accumulation [50,[57][58][59]. In our study, Bna.EPF2 overexpression Brassica transgenic plants showed a lower stomatal conductance and a comparable CO 2 assimilation rate under various CO 2 concentrations and light intensities (Figure 6), which thus resulted in no penalty on the plant growth and seed yield related agronomic traits (Table 1). A possible explanation is that a suitable decrease in the total stomatal pore area (stomatal density and single stomatal pore area) in leaves might have no impact on the total amount of CO 2 necessary for photosynthesis, and the CO 2 supply in these lines was still enough to drive photosynthesis for normal growth under natural field conditions.
In conclusion, we identified the role of the EFP2 ortholog Bna.EPF2 in Brassica, which had a conserved role in stomatal density development and a new, unreported role in stomatal dimension. The overexpression of Bna.EPF2 conferred an enhanced drought tolerance on B. napus plants without a penalty on plant growth or yield. Our results provided an insight into the mechanism of Bna.EPF2 in the regulation of WUE and drought tolerance, and suggested the potential for the manipulation of one gene in the improvement of crop drought tolerance by the alteration of stomatal density and pore size.

Plant Materials and Growth Conditions
Brassica napus (ecotype Westar) and Arabidopsis thaliana (ecotype Col-0) were used in this study. The single T-DNA insertion mutant epf2 (SALK_047918) in Arabidopsis was obtained from the Arabidopsis Biological Resource Center (ABRC).
Arabidopsis and Brassica seeds were surface-sterilized with 75% ethanol for 5 min and 95% ethanol for 30 s, and dried. The seeds were germinated for 7d in a 1/2 MS medium plate at 21 • C after being vernalized at 4 • C for 3 d. The Arabidopsis and Brassica plants were transferred to a compost soil mix (peat moss, perlite, and vermiculite, 1:1:1, v/v/v) and grown in a well-controlled growth chamber or a greenhouse at 22 • C with a 16 h light/8 h dark photoperiod regime.

Sequence Retrieval and Phylogenetic Tree Construction
The amino acid sequences of putative EPFs and EPFLs were obtained from the Arabidopsis Information Resource (TAIR) database (http://www.Arabidopsis.org, 3 March 2023), the Brassica napus genome database (https://www.genoscope.cns.fr/brassicanapus, http://plants.ensembl.org, accessed on 3 March 2023), and the National Center for Biotechnology Information (NCBI) database. The sequences were aligned using MEGA 6.06 software, and a phylogenetic tree was constructed using maximum likelihood method (bootstrap 1000 replications) with default parameters.

Generation of pBna.EPF2::GUS Transgenic Lines and GUS Staining
To generate the pBna.EPF2::GUS expression vector, a 2 kb promoter region upstream of the ATG start codon was amplified by PCR with a primer pair Bna.EPF2-pF and Bna.EPF2-pR using wild type B.napus Westar genomic DNA as a template. The fragment digested by HindIII and BamHI was subcloned into the corresponding restriction sites on the expression vector pLP100 containing a GUS reporter gene [60,61]. The promoter sequence was confirmed by DNA sequencing, and the primers used were listed in Supplementary  Table S1. The pBna.EPF2::GUS construct was transformed into Col-0 using the floral dipping method [62], and transgenic plants were screened on 1/2 MS agar medium containing 50 µg·mL −1 of Kanamycin; the positive transgenic plants were further confirmed by detecting the existence of the GUS reporter gene. The representative T1 lines showing consistent GUS staining were further analyzed for GUS staining at the seedling stage and reproductive stage. At least three independent transgenic lines were analyzed in parallel. For GUS staining, seedlings, flowers and siliques of pBna.EPF2::GUS transgenic Arabidopsis plants were incubated in GUS staining buffer overnight at 37 • C, following the procedure as described [63]. For the observation of expression in stomatal lineage cells, the abaxial leaf epidermal layers, in which mesophyll cells were not in the vicinity, were stained in GUS staining buffer at 37 • C for 2 h. After staining, the seedlings and all tissues were de-colored or rinsed with 75% ethanol several times for GUS observation under a microscope.

Generation of Transgenic Arabidopsis and Brassica napus Plants
The coding sequences (CDSs) of Bna.EPF2 (BnaA09g23140D, Brassica napus AST_PRJEB5043_v1) were amplified from cDNA of Brassica leaves with primers Bna.EPF2-F and Bna.EPF2-R. The PCR products were cloned into the 35S promoter-driving pBI121 binary vector by double digestion with SmalI and XbaI. The 35S::Bna.EPF2 construct was transformed into the Agrobacterium strain GV3101(pMP90) and then into Arabidopsis plants using the floral dipping method [62], and into Brassica napus by Agrobacterium-mediated procedures as described [64]. Transgenic plants were initially selected on 1/2 MS agar medium containing 50 µg·mL −1 of Kanamycin, and further confirmed by the determination of select-marker gene NPTII with PCR. The expression level of positive Bna.EPF2 overexpression lines was analyzed by qPCR analyses, with gene-specific primers listed in Table S1.

Thermal Imaging Analysis
Thermal imaging was performed on 3-week-old Brassica napus plants under normal growth conditions using an infrared camera (FLIR systems; T420), as described [65]. Leaf temperatures were measured in fully expanded first and second true leaves using FLIR Tools + ver. 5.2 software.

Transpiration and WUE Measurements
Photosynthetic and transpiration rates were measured in fully expanded second true leaves of 5-week-old Brassica napus plants using a Li-6400XT Photosynthesis system (LI-6400 Inc., Lincoln, NE, USA). The measurements were applied under the condition of a photon flux density of 300 µmolm −2 s −1 , cuvette flow of 300 mL min −1 , and an ambient CO 2 concentration of 450 ± 5 µmolmol −1 at 21 • C. The rates were recorded every 30 s for 10 min. Rates of photosynthesis (Pn), transpiration (Tr), and stomatal conductance (Gs) were obtained from at least three plants for each genotype per experiment. Instantaneous water use efficiency (iWUE), defined as the ratio of CO 2 assimilated to water lost during transpiration (µmol CO 2 mmol H 2 O -1 ), was calculated from the data of transpiration and photosynthetic rate. Three independent experiments were performed.

Stomatal Density and Stomatal Complex Size Measurement
The stomatal density and stomatal complex size (including stomatal complex length and width, and stomatal pore length, width and area) of the abaxial epidermis of fiveweek-old plants were examined under a light microscope (TS100, Nikon, Tokyo, Japan) described in a previous study [67]. Stomata and pavement cell numbers were counted, and stomatal pore width and length, stomatal pore area, and stomatal complex length and width were measured with Image J 1.42 software (http://rsb.info.nih.gov/ij/, accessed on 3 March 2023). The abaxial epidermis of at least six plants was measured for each genotype per experiment. Experiments were repeated three times. For stomatal density and index measurements in cotyledons, the cotyledon epidermal layers from 15d after germination plants were stained with 10 µg/mL Propidium Iodide for 5min in darkness at room temperature, and then imaged using confocal microscopy (TCS-SP8; Leica, Weztlar, Germany) with 514 nm excitation and 590-630 nm emission.

Drought Stress Assays
Drought stress assays were carried out using two-leaf stage Bna.EPF2-OE lines (each pot containing 16 plants with the same weight of soil and water content). Drought stresses were applied to the plants by withholding watering for 21 days until significant differences in the wilted leaves between different genotypes were observed, and then water was re-supplied for 5 days. Photographs of the plants at these three time points were taken.

Biomass and Yield Trait Measurements
The WT and transgenic Brassica napus plants were cultivated in the experimental farm of the Huazhong Agriculture University, Wuhan, China, in 2021-2022. All the field experiments followed a randomized complete block design with three replications. Each genotype was cultivated to at least six lines. Sufficient irrigation was provided periodically, as needed to supplement rainfall. Routine management was carried out during the whole process of growth. Biomass and yield-related traits, including plant height, length of main inflorescence, number of siliques on the main inflorescence, number of first branch, length per silique, number of seeds per silique, 1000-seed weight, and seed yield per plant, were measured as described previously [68]. All data were collected at the mature stage in May 2022. All weight data were weighed with an electronic balance of ten thousandths, and all length data were recorded in centimeters using a measuring tape.

Statistical Analysis
All data were examined by Student's t-tests. All values were presented as the means ± standard error of the mean (SEM) or standard deviation (SD). A p-value of <0.05 indicated a significant difference between means within WT and overexpression lines.